Australian Journal of Forensic Sciences
Vol. 39, No. 2, December 2007, 107–122
RESEARCH PAPER
STR genotyping of exogenous hair shaft DNA
Kate S. Robertsona*, Dennis McNevinb and James Robertsona
a
Forensic and Technical, Australian Federal Police bForensic Studies,
School of Health Sciences, University of Canberra, Australia
Most hairs found at crime scenes yield low quality and/or low quantities of nuclear
DNA. This DNA is further depleted when stringent hair cleaning procedures are applied
in the laboratory, suggesting that detectable DNA exists exogenously. The phenomenon
of exogenous hair DNA is the subject of this study. DNA was extracted from washed and
unwashed hairs and the resulting ProfilerTM Plus STR genotypes were compared with
those of reference (buccal) swabs from the hair donors. The DNA extraction procedure
involved no prior cleaning of the hair sample and no dissolution of the hair during
digestion, in contrast to standard procedures. The STR genotyping success was measured
by recording the two dominant alleles at each locus and comparing them with the
reference DNA profile. The effect of hair cleanliness was examined by leaving donors’
hair unwashed for periods of 1, 3 and 7 days before sampling. It was found that the
genotyping success for unwashed hair was significantly higher than that for freshly
washed hair, with the majority of clean hair samples producing little or no DNA.
Genotyping success was also lower for donors with cosmetically treated hair compared
with those having untreated hair. Although the quality of STR profiles (i.e. allele
dropout, differential amplification) from hair shafts or telogen hair clubs is reduced
compared with those from other biological sources, the genotypes obtained in this study
may be usable and are certainly discriminating if alternative interpretational methods are
applied.
Keywords: Hair; shaft; telogen; extraction; short tandem repeat (STR); low copy
number (LCN)
1. Introduction
Hair is a biological tissue that can be very useful as forensic trace evidence in criminal
investigations9,8,34. It can be used as evidence to exclude or associate individuals with a
crime scene or object. Until recently microscopic examination of hair morphological
characteristics has been the principal method of hair analysis34. A major limitation of hair
microscopy includes its highly subjective nature, which makes it difficult for the hair
examiner to place a statistical value on a proposed hair ‘match’. These issues have led to a
greater focus on DNA analysis, which can potentially individualise DNA evidence with a
very high statistical certainty. With the advent of the polymerase chain reaction (PCR)29,
highly sensitive nuclear DNA (nuDNA) genotyping systems exist today, allowing
identification of short tandem repeat (STR) microsatellite alleles from minute amounts
of biological material40,42.
*Corresponding author. Email: [email protected]
ISSN 0045-0618 print/ISSN 1834-562X online
ß 2007 Australian Academy of Forensic Sciences
DOI: 10.1080/00450610701650096
http://www.informaworld.com
108
K. S. Robertson et al.
Currently, nuclear DNA analysis is predominantly conducted only on the roots of
hairs in the active growth (anagen) phase rather than on hair shaft or hairs in the resting
(telogen) phase. While anagen phase hair has metabolically and mitotically active root and
follicle material that are amenable to DNA typing, hair shaft and telogen hair clubs are
fully keratinised containing only very small amounts of DNA that are thought to be of a
very degraded nature25,36,39. This is problematic for forensic scientists as it is the fully
keratinised telogen phase hairs that are naturally shed and comprise the majority of
evidentiary hairs found at crime scenes4,11.
As a result there has been considerable focus on mitochondrial DNA (mtDNA)
analysis, which has been more successful than nuDNA analysis20,21,23,28,31 due to mtDNA
existing in much higher copy numbers within a cell.43 However, mtDNA analysis has
several limitations26, a significant one being that it is maternally inherited and thus
mtDNA profiles cannot individuate between maternal relatives. Hence the establishment
of a successful and reliable technique for the typing of nuDNA from keratinised hair
would be of great value to the forensic community.
Recent research into the improvement of STR typing of DNA from keratinised hair
has mainly focused on post-extraction procedures, including techniques such as reduced
volume PCR (RV-PCR), extended PCR cycles, nested PCR and the use of redesigned PCR
primers that generate shortened PCR products7,12,13,15,16,18. However, it is not clear
whether nuDNA actually persists endogenously within the hair shaft or the telogen hair
club26. McNevin et al. 27 found that there was a greater chance of obtaining STR alleles
that corresponded with donors’ buccal swabs (‘consensus’ alleles) from single hair shafts
not cleaned prior to extraction rather than from cleaned hair shafts, where cleaning had
involved sequential washes with SDS, dH2O and alcohol. They also showed that the most
effective procedure for obtaining consensus alleles from hair shaft involved only rinses
with water and a Tris–HCl buffer which did not dissolve the hair27. A better result was not
obtained by dissolution of the hair in a lysis buffer containing commonly used detergents,
proteinase K and reducing agent. The observations that the degradation of the keratin hair
structure has little effect on nuDNA quantity and quality, and that pre-cleaning the hair
decreases the chance of obtaining nuDNA, provides evidence that recoverable nuDNA
resides towards the hair exterior or is exogenous. Indeed, there is considerable evidence
that nuDNA exists in the cuticle layer of the hair shaft19,24,30,35.
Using an extraction method involving simple leaching of exogenous nuDNA from
hair, McNevin et al. 27 found that STR genotyping success was highly dependent on the
donor. This is consistent with the proposition that exogenous DNA is more vulnerable to
the environmental influences on the hair. From a forensic perspective, if the majority of
recoverable nuDNA is indeed exogenous, a better understanding of the factors or
conditions that can affect the presence of nuDNA on hair, and thus the success of STR
genotyping, is critical.
The aims of this study were to identify factors that may affect the presence of
exogenous DNA on hair shaft, with a major focus on hair cleanliness. The effect of hair
washing on the ability to obtain a STR profile from hair shaft is investigated by
comparison of DNA profiles produced from clean hair and dirty hair from a number of
donors. It is hypothesised that if hair shaft DNA is exogenous, and therefore more
vulnerable to environmental influence, then hair washing will remove the recoverable
exogenous DNA and consequently there will be a marked difference between the ability to
obtain a profile from freshly washed hair and dirty hair. An alternative STR profile
interpretation method is recommended for this type of low copy number (LCN)
genotyping.
Australian Journal of Forensic Sciences
2.
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Materials and methods
2.1 Sampling procedures
A total of 29 volunteers were recruited to the study in accordance with the University of
Canberra Human Ethics Manual (2007)22. The participants consisted of 8 males and 21
females and ranged in age from 22 to 53 years. They each collected a minimum of 15
individual hairs directly after washing their hair with supplied shampoo (De LorenzoÕ ,
Hair and Cosmetic Research Pty. Ltd., Silverwater, Australia) and then another minimum
of 15 hairs a number of days later without further shampoo washing (washing with water
only was permitted). A clean comb was provided each time hair was sampled. The
majority of participants fell into three groups, those that left their hair unwashed for 1 day
(6 participants), 3 days (12), or 7 days (11). Hairs were self-sampled by participants either
by running a comb or gloved hands through the hair. Hair samples were stored at room
temperature in folded paper inside sealable plastic bags until examined.
To assist in identification of other factors that may influence genotyping success, each
participant completed a questionnaire, which provided information on the features,
condition and recent history of their hair and how it was treated during the period between
collection of the clean and dirty hair samples (factors identified in the Questionnaire are
included in Table 1).
A buccal (cheek) swab (Medical Wire & Equipment Co. (Bath) Ltd., Corsham, UK)
was obtained from each participant in order to produce a reference DNA profile. The
buccal swab was stored in a paper envelope at 4 C until required for DNA extraction.
2.2 Hair sample preparation
Eight hairs were used from each clean and dirty hair sample supplied by the participants.
The hairs were examined under a stereomicroscope (40 resolution) to identify the hair
root, then for each hair approximately 1.5 cm was cut from the proximal end and
discarded. A further 2.5 (0.3) cm segment from each of the eight hairs was then cut from
the proximal end and placed in a sterile 1.5 ml tube, yielding an equivalent total of 20 cm of
hair in each sample tube. Hair sample preparation was conducted in a separate room to
reference swabs. All equipment used was washed with 20% bleach and 70% ethanol
solutions before processing each hair sample to minimise the risk of contamination.
2.3 Sample processing
As a precaution against extraneous contamination, DNA extraction and DNA
amplification were performed in different rooms. All samples were handled in a
presterilised biological safety (laminar flow) cabinet with latex gloves, hair net, and
facemask. Hair samples were processed separately from reference samples with
approximately 1 month between reference and hair sample processing. The laminar flow
cabinet and all equipment were decontaminated between the processing of each sample
batch. All reagents and chemicals used were of Analytical Reagent (AR) grade or
molecular biology grade as appropriate.
2.4 DNA extraction
DNA from the clean and dirty hair samples was extracted using a phenol-chloroform
extraction method optimised for the extraction of exogenous hair DNA, as described by
McNevin et al. 27 This protocol involved no cleaning of the hair samples prior to extraction
and no dissolution of the hair during the extraction process. Each sample was soaked
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K. S. Robertson et al.
Table 1. Demographic information for the 29 participants obtained from the
questionnaire.
Sex
Males
Females
Age
18–35 years
36–53 years
Ethnic Origin
NW European
S European
NW European, S European
NW European, African
NW European, Oceanian (Maori)
Hair Type
Wavy hair
Straight hair
Curly hair
Hair Length
Short hair
Short-medium
Medium
Long
Treated and Natural Hair (Hair used in experiments)
Treated hair
Permanent dye and/or bleached
Permanent foils
Semi-permanent
Temporary dye rinse
Natural hair
Blonde
Blonde-Brown
Brown
Black
Red
Grey
Use of Supplied Conditioner
Conditioner used
No conditioner used
Hair Products used during Experiment
No hair product used
Hair product used
Conditioning & smoothing products
Hairspray, wax, putty, gel & curling crème
Conditions Hair is Exposed to
No specific conditions
Specific conditions
Chorinated water regularly, during experiment
Chorinated water regularly, but not during
experiment
Paint fumes and dust, everyday
8
21
18
11
24
2
1
1
1
10
17
2
15
3
7
4
9
(6)
(1)
(1)
(1)
20
(4)
(1)
(11)
(1)
(0)
(3)
27
2
16
13
(2)
(11)
25
4
(2)
(1)
(1)
(continued)
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Australian Journal of Forensic Sciences
Table 1. Continued.
Oiliness of hair
Dry
Normal
Oily
Combination
Usual Regularity of Hair Washing
Every 1 day
Every 2 days
Every 3 days
Every 4 to 7 days
Greater than every 7 days
4
16
7
2
11
8
5
4
1
overnight in a simple digestion buffer containing 10 mM Tris–HCl, 10 mM EDTA, 2%
TweenÕ 20 and 100 mM NaCl. After extraction with phenol:chloroform:isoamyl alcohol
(25:24:1) and chloroform each sample was subjected to centrifugal ultrafiltration using
MicroconÕ YM-30 centrifugal filter devices (Millipore Corporation, Bedford, USA). The
resulting 100 ml extracts were stored at 4 C until quantitation.
DNA from the buccal swabs was extracted using ChelexÕ 100 Resin (Bio-rad,
Hercules, CA) according to standard procedures41.
2.5 DNA quantitation
The amount of extracted DNA was estimated via real-time PCR as described by
McNevin et al.27
2.6 DNA amplification
DNA extracts from buccal swabs and hair samples were genotyped against AmpF‘STRÕ
Profiler PlusTM STR loci (Applied Biosystems, Foster City, CA). Extracted DNA was
amplified in a GeneAmpÕ 9600 thermal cycler (Applied Biosystems) in a 50 ml volume
according to the AmpF‘STRÕ Profiler PlusTM protocol5 except that, for hair samples, 34
PCR cycles (rather than the standard 28 cycles) were employed and a 100 dilution of
AmpF‘STRÕ Control DNA 9947A (to make final concentration of 1 pg/ml) was used as a
positive control because of the extra 6 PCR cycles. PCR product (1.5 ml) was then added
to 14.4 ml of Hi-DiTM deionised formamide (Applied Biosystems) and 0.6 ml of
GeneScanÕ -500 [ROX]TM internal standard (Applied Biosystems) and then separated
into STR alleles via capillary electrophoresis on an ABI PrismÕ 3100 Genetic Analyser
(Applied Biosystems) under the following conditions: 36 cm array; POP-4TM sieving
polymer (Applied Biosystems); filter set F; 10 s, 5 kV injections; 60 C, 15 kV runs.
Electropherograms were generated using GeneScanÕ Analysis Software2. STR genotypes
were determined in the GenotyperÕ Software1 environment.
2.7 Interpretation of STR profiles
Allele peaks (homozygous and heterozygous) that were less than 100 RFU (relative
fluorescence units) were not recorded, in accordance with standard Australian Federal
Police procedures3. Stutter artefacts were interpreted according to the AmpF‘STRÕ
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K. S. Robertson et al.
Profiler PlusTM PCR Amplification User’s Manual (2000)5 and, if present below the
maximum expected stutter percentage for the locus in question, they were ignored.
Due to a relatively high incidence of PCR artefacts and possible contaminating DNA
(‘non-consensus’ alleles) in the hair extract profiles, only the two dominant alleles (two
highest peaks as measured by RFU) at each locus were recorded. The profile resulting
from the application of this rule was termed a ‘primary profile’.
The genotypes for the hair extracts were compared with their corresponding reference
genotypes obtained from buccal swabs. The STR genotyping success was determined as
the number of dominant alleles that matched the reference genotype (a maximum of 20). If
one of the two dominant peaks at a locus in a sample DNA profile matched a homozygous
reference allele for that particular locus, then both homozygous alleles were considered to
be present.
2.8 Statistical analysis
Non-parametric statistical analyses were conducted on the data produced due to very
skewed data distributions. All statistical analysis was performed using SPSSÕ Analytical
Software for WindowsÕ Version 11.537. Only the primary profiles were considered in the
statistical calculations.
3. Results
The demographic and hair information obtained from the questionnaires issued to
participants is depicted in Table 1. The participants ranged in age from 22 to 53 years, with
only 27.6% (8) being male and the majority being of a North Western European ethnic
origin (82.8%). Two-thirds of the participants had naturally coloured hair (69.0%), which
ranged in colour from blonde to black. Treated hair consisted of temporarily dyed (colour
rinses), bleached, permanently dyed (including foils) and semi-permanently dyed hair.
The quantities of DNA in the hair shaft extracts, as estimated by real-time PCR, were
very low. Only six samples contained detectable DNA, ranging from 1.6 pg ml1 to
5.1 pg ml1. The capacity to measure the DNA concentration was not indicative of the
ability to obtain a DNA profile, as several samples that produced a full profile did not
produce a measurable DNA concentration by real-time PCR. The quantitation method,
based on the Alu transposon in the human genome27, may not be appropriate for the
analysis of very low levels of DNA.
Contaminating alleles appeared in two of the extraction controls and one of the
negative controls. The average number of contaminating alleles in all negative and
extraction controls analysed was 0.33 alleles per control. There was no consistency as to
which contaminating alleles were present in the controls and these alleles were not present
in hair profiles unless they corresponded with reference profiles. When genotyping of the
controls was repeated, contaminating alleles disappeared. These observations suggest that
the contamination of the controls was a random event and not due to contamination of the
reagents used.
Only 42 of the 58 hair extracts produced DNA profiles (72.4%), 15 of the clean hair
extracts and 27 of the dirty hair extracts. A total of 87 and 289 consensus alleles were
observed in the clean hair and dirty hair primary profiles, respectively. Analysis of only the
dominant alleles (primary profile) did not affect participants’ genotyping success except in
one case where it was reduced by one, dropping the total consensus allele count for all
dirty hair profiles from 290 to 289. However, it did reduce the total number of
Australian Journal of Forensic Sciences
113
non-consensus alleles in the profiles dramatically, from a total of 30 when all observable
peaks were considered to a total of 16 when only the dominant alleles (primary profile)
were considered. Although the number of non-consensus alleles was higher in the dirty
hair primary profiles (12) than the clean hair primary profiles (4), the difference was not
significant (Sign test, p ¼ 0.114).
Figure 1 shows the distribution of the number of consensus alleles in the clean and
dirty hair profiles. The number of consensus alleles in the clean hair profiles varied from
0 to 19 with just under half the participants producing no profile at all (48.3%). There were
two outliers that produced 85% and 95% of their full consensus profile in their clean hair
sample (17 and 19 alleles, respectively). In contrast, the dirty hair data had a much less
skewed distribution with 5 (17.2%) samples producing full profiles, 22 (75.9%) producing
partial profiles and only 2 (6.9%) producing no profile. A sign test was used to compare
the median number of consensus alleles in the clean and dirty hair profiles. The clean hair
(a) 16
Number of participants
14
12
10
8
6
4
2
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Number of consensus alleles in clean hair primary profile
(b) 16
Number of participants
14
12
10
8
6
4
2
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Number of consensus alleles in clean hair primary profile
Figure 1. The distribution of the number of consensus alleles in the (a) clean hair primary profiles
and (b) dirty hair primary profiles. The arrows highlight the two participants who produced 85%
and 95% of their full profile.
114
K. S. Robertson et al.
median (1.0) and the dirty hair median (9.0) were found to be significantly different
(p ¼ 0.000).
Table 2 shows the number of consensus alleles observed in the clean and dirty profiles
of each group. For the 3-day and 7-day group data, the dirty hair median was significantly
higher than the clean hair median (Sign test, p ¼ 0.000 and p ¼ 0.006, respectively);
however, for the 1-day group the clean and dirty hair median were not significantly
different (Sign test, p ¼ 0.063). Although there was no significant difference between the
dirty hair data of the 3-day and 7-day groups (p ¼ 0.440), the number of consensus alleles
detected in the participants dirty hair profiles varied greatly, ranging from 1 to 20 alleles
(data not shown). Hair treatment was investigated as a possible contributing factor to this
variation in genotyping success. The data of the participants in the 3-day and 7-day groups
were pooled into one set as they were not significantly different, which allowed for a
greater population sample to be studied (23 participants), and then separated according to
whether they had natural hair or treated hair (Figure 2). Treated hair was considered to be
hair that had been dyed (permanent, semi-permanent or rinse treatments), bleached or
contained foils (see Table 1). To take into account the presence of re-growth, participants
who stated that their hair had been treated 4 months ago or longer were classified as
‘natural’. This limit was based on the estimation that hair grows approximately 1 cm every
Table 2. The total number of consensus alleles observed in the clean and dirty profiles for each
group and the median number of consensus alleles in the clean and dirty hair primary profiles for
each group.
Clean Hairc
Dirty Hair
Groupa
Nb
Total Alleles Median
Median
Total Alleles Median
Median
1-day
3-days
7-days
6
12
11
4
37
46
0
1
2
29
142
118
2
11
9
a
Participants grouped according to the day they collected their dirty hair sample.
Number of participants in each group.
c
Represents the sample taken at day 0 for each group.
b
Number of consensus alleles
20
15
10
5
0
Treated
Natural
Figure 2. Consensus allele counts for the 23 participants’ dirty hair profiles (3 days dirty),
grouped according to whether the profiles were produced from natural or treated hair. The
grey boxes represent the middle 50% of values and each whisker represents 25% of the values to
either side. The black lines represent the medians.
Australian Journal of Forensic Sciences
115
month17 and the hair samples used for DNA analysis in this study were obtained from
within the first 4 cm of the proximal end of each hair. A Mann–Whitney Test was used to
compare the two independent data sets and showed that the two groups were significantly
different (p ¼ 0.011). Similar analyses were conducted with regard to the use of hair
product and participant hair type (curly/straight/wavy), however no significant differences
were found between these groups and the number of consensus alleles in participant dirty
hair profiles (data not shown).
There are many other factors that may have some influence on the STR genotyping
success of hair shaft DNA. Table 1 contains information on several characteristics that
may be potential contributing factors. For example, a participant’s ethnic origin, hair type,
oiliness of the hair or the use of hair products may affect the ability to obtain DNA from
their hair shaft. The number of participants involved in this study does not provide enough
statistical power to measure many of these factors.
It should be noted that the influence of extraneous variables during hair sampling
might interfere with drawing clear conclusions from the data obtained. Steps taken to
reduce error variance due to these variables included treatment of subjects as similarly as
possible, consistency in presentation of instructions to each participant and use of
standard sampling and laboratory protocols. Even so, subjects and their living
environments differ from one another in innumerable and unidentifiable ways, and the
possibility that participants deviated from the instructions provided cannot be discounted.
Several features were observed that affected the quality of the hair extract profiles.
Differential amplification of heterozygote alleles resulting in unbalanced peak heights was
present in most of the profiles and was also present in the 100 diluted positive control
(Figure 3a). Another common observation was that homozygous and heterozygous alleles
were not present in their usual proportions, with homozygote peaks sometimes being
smaller than heterozygote peaks (Figure 3b and c).
Allelic dropout was also a very common observation in the partial profiles obtained
(Figure 3c). Figure 3d is a further example of allelic dropout, although this time with a
non-consensus contaminating allele present as well, another common feature observed.
Contaminating ‘non-consensus’ alleles were present in both the clean and dirty hair
samples. There was no consistency except possibly for allele 14 of the D8S1179 locus and
allele 8 of the D1S317 locus, both of which appeared in two different profiles (but not in
the controls). The number of non-consensus alleles did not increase with the number of
consensus alleles in the participants’ dirty hair profiles (rs ¼ 0.082, p ¼ 0.661).
Although most of the peaks corresponding to non-consensus alleles were smaller than
the peaks of consensus alleles, as in Figure 3d, in two cases the contaminating allele had a
higher RFU value than the consensus allele (Figure 3e and f). In every hair profile but one,
all consensus alleles present at a locus were included in the two dominant peaks of the
primary profile. In the one exception (Figure 3f), the contaminating allele was bigger than
one of the consensus heterozygous alleles, which excluded that heterozygous allele from
the primary profile.
The measure of STR genotyping success so far mentioned does not indicate the
accuracy of the profile with respect to the presence of the non-consensus alleles. The degree
of reliability of a genotype is defined here as the ratio of the number of non-consensus
alleles to the number of consensus alleles present in the DNA profile. Table 3 shows the
genotype reliability for the clean hair and dirty hair primary profiles for each of the
participants. Even though the total ratio value is lower for clean hair (1.72), indicating that
obtaining a genotype from clean hair is more reliable, the difference between the total ratio
values of clean and dirty hair is not significant (Sign test, p ¼ 0.194). In addition, there is
116
K. S. Robertson et al.
Figure 3. Profiler PlusTM electropherograms of several STR loci depicting cases of reduced profile
quality. All were obtained from dirty hair except 3 c. (a) Electropherogram of the D21S11 locus,
showing differential amplification of heterozygous alleles. (b) Electropherogram of the D13S317 and
D7S820 loci, showing disproportional peak heights of heterozygote and homozygote loci, in which
the homozygous peak is approximately the same as the heterozygous peaks. (c) Electropherogram of
the D3S1358 and vWA loci, illustrating another case of disproportional peak heights of heterozygote
and homozygote loci in addition to allele drop out. The peak at the vWA locus corresponds to a
heterozygote locus although only one heterozygous allele was detected. The arrow indicates that the
homozygote peak at the D3S1358 locus is approximately half the size of the heterozygous peak.
(d) Electropherogram of locus D3S1358 for a heterozygote individual in which allele dropout has
occurred resulting in detection of only one heterozygous allele (allele 14). The arrow highlights the
presence of a smaller contaminating allele (allele 15). (e) Electropherogram of the D21S11 locus for a
heterozygote individual in which allele dropout has occurred resulting in detection of only one
heterozygous allele (allele 30). The arrow highlights the presence of a larger contaminating allele
(allele 29). (f) Electropherogram of the D5S818 locus for a heterozygote individual, in which a
contaminating peak (allele 14) has been detected in greater amount than one of the heterozygous
peaks. (g) Electropherogram of the D5S818 locus for a homozygote individual, in which a
contaminating allele (allele 11) appears alongside the taller homozygote peak. (h) Electropherogram
of the D5S818 locus showing base pair ladder and allele designations. The arrow indicates the
presence of a short non-consensus peak that is four base pairs smaller in size than the heterozygous
allele 11, which may be the result of over-amplification of a stutter artefact.
no significant difference between the non-consensus-to-consensus ratios for the dirty hair
samples of the 3- and 7-day groups (Mann–Whitney U test, p ¼ 0.651). The 1-day group
was excluded from this analysis due to the small sample size.
Increased amplification of stutter artefacts beyond the expected maximum loci stutter
peak percentages as expressed in the AmpF‘STRÕ Profiler PlusTM PCR Amplification
Users Manual (2000)5 may explain the presence of some non-consensus alleles: one
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Australian Journal of Forensic Sciences
Table 3. Ratio of the number of non-consensus alleles to the number of
consensus alleles in the clean hair and dirty hair primary profiles for each
participant.
N-C/Cb
Days
Code
Dirtya
Clean Hair
1
2
3
4
5
6
Bx
Ex
Ix
Nx
Aax
Afx
1
1
1
1
1
1
–
–
–
–
–
7
8
9
10
11
12
13
14
15
16
17
18
Ax
Fx
Hx
Kx
Ox
Qx
Rx
Sx
Vx
Wx
Xx
Zx
3
3
3
3
3
3
3
3
3
3
3
3
0
0
–
0
0
1.00
0
0.14
0
0.17
0
0
0
0
0.44
0
0
0
19
20
21
22
23
24
25
26
27
28
29
Cx
Dx
Jx
Lx
Px
Tx
Yx
Abx
Adx
Aex
Agx
7
7
7
7
7
7
7
7
7
7
7
–
–
0.17
0
0
0
–
0.50
0.05
0
–
0
0.13
0.07
0
0.13
0
0.33
0
0
0.06
0
N/A
N/A
1.72
0
1.97
0
Participant
Total
Median
0
–
–
–
0
–
0
Dirty Hair
0
0
–
–
0.50
0
a
Indicates the number of days after washing and before sampling.
The ratio of the number of non-consensus alleles to consensus alleles in a profile.
A dash indicates that no profile was obtained. A value of ‘0’ indicates there were
no non-consensus alleles observed (most reliable genotype).
b
example is observed in Figure 3h. The presence of these potentially over-amplified stutter
peaks did not have any effect on the respective primary profiles obtained.
4. Discussion
Our results show that it is possible to obtain discriminating STR profiles from hair shaft
that does not undergo a cleaning step prior to extraction and is soaked in a simple digest
buffer, which does not dissolve the hair. Genotyping success of hair shaft DNA was
dependent on the hair donor, as found by McNevin et al. 27 The genotyping success of the
(dirty) hair extracts varied extremely between individuals, ranging from 100% success with
118
K. S. Robertson et al.
all of the possible 20 alleles present (17.2% of participants) to no success with no alleles
present (6.9% of participants).
A comparison of the DNA profiles produced from each participant’s clean and dirty
hair samples showed that there was a significant increase in the genotyping success for
dirty hair. The increase in the DNA detected indicates that the hair has acquired DNA
during the period of time between shampooing and sampling of the dirty hair, which
suggests that the additional DNA was most likely deposited onto the hair and exists
exogenously. The significantly lower success in STR genotyping of freshly washed hair
would indicate that hair washing removes some of the detectable DNA that exists
exogenously on the hair shaft. This has been shown in several other studies30,35. Schreiber
et al. 35 found that washing hair with an aqueous solution of the detergent SDS drastically
reduced the DNA yield obtained from the hair. Heywood et al. 19 showed that washing
hairs with a diluted alkaline shampoo (12% sodium lauryl ethyl sulfate, 2% tegobetaine)
resulted in detectable DNA in the wash solution after 20 sequential washes.
Although significantly lower, consensus alleles were still detected in several of the clean
hair profiles (51.7% of participants). Indeed, two individuals produced 85% and 95%
(17 and 19 alleles, respectively) of their full consensus profile in their clean hair sample.
This only serves to illustrate the donor-variability of genotyping success. If endogenous
DNA exists at all in hair shaft, it is most likely to be found in the cuticle cells19,24,30 and
thus may be accessible to a digestion buffer.
According to our results, if (telogen phase) hair evidence found at a crime scene was
freshly washed or had been unwashed for only one day, it most likely would not yield a
discriminating STR profile. The chance of obtaining a useful DNA profile increases if the
hair has been unwashed for 3–7 days. However, although all samples produced DNA in
these two groups, the majority of profiles produced at 3 and 7 days were only partial
profiles (82.6% or 19 participants). Only 17.4% of the profiles (4 participants) had the
maximum 20 consensus alleles providing the highest discriminatory power possible using
the Profiler PlusTM genotyping system. Due to the inability to obtain a full DNA profile
from the majority of participants, it would be desirable to conduct further studies to
determine if the genotyping success for hair would increase further if the hair were left
dirty for longer than 7 days. From a forensic investigative perspective, this may be
pertinent as hair evidence often presents in a relatively dirty state33.
In contrast to the above results, one participant whose hair was sampled one day after
washing produced a full DNA profile (the median number of alleles for similar samples
was 1.5). This hair sample collected as much exogenous DNA as hairs that were left
unwashed for 3 and 7 days. A feature that may be relevant in distinguishing this
participant from others is the participant’s use of a ‘hair putty’ product.
The genotyping success for untreated hair was significantly higher than that for treated
hair. Other studies have reported similar findings. Heywood et al. 19 showed that the
amount of extractable DNA was significantly reduced after permanent colour treatment of
hair (with commercial hair colour treatment), while McNevin et al. 27 found that bleaching
hair (with commercial hair bleach) similarly reduced the amount of recoverable DNA. The
correlation between hair treatment and recoverable DNA may be due to alteration of the
hair surface (physical or chemical), which may either damage the cuticle cells or decrease
the ability of DNA to adhere to them. It has been widely documented that treatment of
hair with cosmetic products can induce limited temporary surface changes or more
extensive, permanent whole-fibre changes to the basic morphology and chemistry of
the hair10,32.
Australian Journal of Forensic Sciences
119
Low copy number (LCN) PCR inevitably results in a reduction in the quality of the
hair extract profiles obtained and these have important implications for forensic casework.
Increased stutter artefacts, allele dropout and unbalanced heterozygosity were regularly
observed and are thought to be the result of stochastic variation when pipetting very dilute
DNA extracts14,38. Standard interpretation methods, such as use of peak size and
proportion, for allocation of genotypes to DNA profiles are probably not appropriate
when very low quantities of DNA are extracted from hair. The amplification of
laboratory-based random contaminating alleles, otherwise known as ‘allele drop-in’, was
detected in two extraction controls and a negative control and may possibly be responsible
for several non-consensus alleles in the hair profiles. This is a common feature of LCN
DNA amplification where 34 PCR cycles (or more) are used. Theoretically, this method is
sensitive enough to detect the equivalent of a single copy of DNA15. A sterile workplace
and the use of DNA-free reagents and instruments are essential for the analysis of DNA
from keratinised hair.
We have applied an alternative STR profiling strategy for assigning genotypes to LCN
DNA which involves the examination of a ‘primary profile’ comprising the two dominant
alleles at each locus. There will always be a danger here that a contaminating allele will
exclude an allele derived from the hair donor. This occurred in one case in our results
(Figure 3f). However, we have demonstrated that with use of this method, the effect of low
levels of random contaminating alleles and enhanced stutter peaks can be reduced to a
minimum.
Several areas of further improvement were identified that must first be addressed
before advocating this method for operational use. It is highly recommended that replicate
analyses are conducted with LCN DNA to identify random contaminating or ‘drop-in’
alleles and achieve unambiguous genotypes15,38. It may be necessary to quantify the
probability of random contamination by performing numerous extraction and negative
controls. Identification of non-reproducible random ‘drop-in’ contamination would help
to prevent misinterpretation of an individual’s primary profile.
In this study, 8 2.5 cm proximal hair segments (20 cm in total) were combined in each
sample so that differences between long and short hair were minimised. Obviously,
casework will provide a range of hair lengths, as well as single hairs, and therefore it would
be ideal to conduct additional tests using more realistic scenarios. Also, because buccal
swabs were available from hair donors, homozygous genotypes could be identified here
where this would not be possible in casework. Identification of homozygosity is a
limitation of this and other approaches to LCN DNA genotyping. However, there are
methods available for interpretation of these profiles14,15,38.
At present, this and other LCN strategies may not be tenable in court. However, they
are very useful in focusing forensic investigations and their power for inclusion and
exclusion of suspects should not be discounted.
5. Conclusion
This study has provided further evidence for the existence of exogenous hair shaft DNA
and has shown that it is quite possible to obtain highly discriminating STR profiles from
hair shafts. Factors affecting the ability to obtain a profile have also been identified. The
chance of obtaining a native STR profile is much greater when hair has not been washed
for 3 days or more. Given the variability between individuals in obtaining an STR profile
and the issues concerning poor profile quality, the STR genotyping of keratinised hair
120
K. S. Robertson et al.
remains problematic. In this study, an alternative strategy for the assignment of genotypes
when using LCN techniques has been proposed, while the limitations of this strategy have
been identified.
Analysing nuDNA from hairs continues to attract interest from several groups of
researchers. Most recently Anslinger et al.6 reported on the analysis of 96 hairs taken from
routine casework, using two short amplicon or mini STR kits and real-time PCR as a
screening method for hair selection. Sixty-five of the hairs tested gave no detectable DNA.
All of these were telogen hairs. Several of the successful hairs had visible cellular (sheath)
material present and the root type in others was unknown. Even with these hairs the
overall success rate was just over 20%.
Hence, it is clear that success in obtaining nuDNA from teleogen hairs remains
technologically challenging. We believe that the hair should be viewed as a substrate for
exogenous DNA and that any protocol for the selection of hairs should be based on a
knowledge of the donor history where possible. nuDNA testing of telogen hairs cannot yet
be considered a routine technique but it is within our grasp. We predict limited
applications of this approach in the next few years after further study of variables which
might affect potential successful typing and further advances in short amplicon analytical
approaches.
Acknowledgments
The authors wish to thank Jennelle Kyd for valuable advice on experimental design for
this study.
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